Synthesis of oligomeric epicatechin and catechin-derived procyanidins

ABSTRACT

Various processes are disclosed for preparing procyanidin oligomers having (4,8)-interflavan linkages. In an improved process, a tetra-O-protected-epicatechin or -catechin monomer or oligomer is coupled with a protected, C-4 alkoxy-activated-epicatechin or -catechin monomer in the presence of an acidic clay instead of a Lewis acid. In a second process, a 5,7,3′,4′-tetra-O-protected or preferably penta-O-protected-epicatechin or -catechin monomer or oligomer is reacted with a tetra-O-protected or preferably penta-O-protected-epicatechin or -catechin monomer having a thio activating group at the C-4 position; the coupling is carried out in the presence of silver tetrafluoroborate. In third process, two molecules of a penta-O-protected-epicatechin or -catechin monomer activated with a 2-(benzothiazolyl)thio group at the C-4 position are self-condensed in the presence of silver tetrafluoroborate. An improved two-step process for preparing a C-4 alkoxy activated tetra-O-benzyl-protected, 8-bromo-blocked-epicatechin or -catechin monomer is also provided. The use of naturally-derived and synthetically-prepared procyanidin (4β,8) 4 -pentamers to treat cancer is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT/US03/31375, filed Oct. 2, 2003,which is a continuation in part of Ser. No. 10/658,241 filed Sep. 9,2003, now U.S. Pat. No. 7,067,679, which is a utility application basedon provisional application Ser. No. 60/415,616 filed Oct. 2, 2002.

BACKGROUND OF THE INVENTION

Condensed tannins (proanthocyanidins) are widespread in the plantkingdom, form part of the human diet, and display multiple biologicalactivities that render them significant to health. Procyanidins haveattracted a great deal of recent attention in the fields of nutrition,medicine and health due to their wide range of potentially significantbiological activities. There is a growing body of evidence suggestingthat these compounds act as potent antioxidants in vitro, ex vivo and invivo and may thus alter the pathophysiology of imbalances orperturbations of free radical and/or oxidatively driven processes inmany diseases or directly interfere with many cellular processes. SeeNijveldt, R. J. et al., Am. J. Clin. Nutr. 2001, 74, 418. Initialobservations also have shown that procyanidin-rich fractions extractedfrom defatted cocoa beans elicited in vitro growth inhibition in severalhuman cancer cell lines. See U.S. Pat. No. 5,554,645 issued Sep. 10,1996 to L. J. Romanczyk, Jr. et al., the disclosure of which isincorporated by reference.

Isolation, separation, purification, and identification methods havebeen established for the recovery of a range of procyanidin oligomersfor comparative in vitro and in vivo assessment of biologicalactivities, and currently some oligomers can be synthesized usingtime-consuming methods. For instance, previous attempts to couplemonomeric units in free phenolic form using mineral acid as the catalystin aqueous media have met with limited success. The yields were low, thereactions proceeded with poor selectivity, and the oligomers weredifficult to isolate. See Steynberg, P. J., et al., Tetrahedron, 1998,54, 8153–8158. An overview of the shortcomings is set out below.

The benzylated monomer was prepared from the free monomer using benzylbromide in combination with potassium carbonate (K₂CO₃) and dimethylformamide (DMF). See Kawamoto, H. et al., Mokuzai Gakkashi, 1991, 37,741–747. The yield, however, was only about 40%. In addition, competingC-benzylation leads to a mixture of products, which make isolation ofthe benzyl-protected target monomer more difficult. Also, partialracemization of (+)-catechin at both the C-2 and C-3 positions wasobserved (see Pierre, M.-C. et al., Tetrahedron Letters, 1997, 38,5639–5642).

Two primary methods for oxidative functionalization are taught in theliterature. See Betts, M. J. et al., J. Chem. Soc., C, 1969, 1178 andSteenkamp, J. A., et al., Tetrahedron Lett., 1985, 3045–3048. In theolder method, protected (+)-catechin was treated with lead tetraacetate(LTA) in benzene to produce the 4β-acetoxy derivative which was thensuccessfully hydrolyzed to the 3,4-diol. Flavan-3,4-diols are incipientelectrophiles in the biomimetic synthesis of procyanidins. The majordrawback in the oxidative functionalization of the prochiral benzylicposition was a low yield (30–36%) of the acetate during the leadtetraacetate (LTA) oxidation. The more recent method of oxidativelyfunctionalizing the C-4 position relies on the use of2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In this method, theprotected monomer was treated with DDQ in methanol. This allowsintroduction of a methoxyl group at the C-4 position in astereoselective manner. The yield was about 40–50%

There are a number of reports on the coupling reaction between monomersand their 3,4-diols in aqueous acid. These methods are unsatisfactorybecause of low yields, lack of selectivity, and difficulty in thepurification from aqueous media. See Kawamoto, H. et al., J. Wood Chem.Technol., 1989, 9, 35–52 who report the titanium tetrachloride (TiCl₄)mediated coupling between 4-hydroxy-tetra-O-benzyl-(+)-catechin and 5equivalents (eq.) of tetra-O-benzyl-(+)-catechin to produce a 3:2mixture of 4α,8 and 4β,8 catechin dimers. This coupling leads to the4β,8-dimer together with higher oligomers in yields that decrease withthe increasing molecular mass of the oligomer.

Using a 2,3-cis-3,4-trans-flavan-3,4-diol, procyanidins B₂ and B₅derivatives were synthesized. The diol was prepared by the acyloxylationof the C-4 benzylic function of a (−)-epicatechin tetramethyl ether withlead tetraacetate in a benzene solution. This oxidativefunctionalization of the C-4 position of the methyl-protectedepicatechin monomer was improved by using2,3-dichloro-5,6-dicyano-1,4-benzoquinone(DDQ) in methanol to introducea methoxyl group at the C-4 position. The protected C-4 methoxy monomerwas used in the synthesis of 4,8-linked linear procyanidin oligomers upto the trimers. See Steenkamp et al., Tetrahedron. Lett. 1985, 26,3045–3048.

Procyanidin oligomers were prepared using a protected epicatechin orcatechin monomer having, as a C-4 substituent, a C₂–C₆ alkoxy grouphaving a terminal hydroxy group such as a 2-hydroxyethoxy group. Theprotecting groups used are those that do not deactivate the A ring ofthe monomer, e.g., benzyl protecting groups. See Kozikowski, A. P. etal. J. Org. Chem. 2000, 65, 5371–5381 and U.S. Pat. No. 6,207,842(issued Mar. 27, 2001 to Romanczyk, L. J. et al.). The C-4 derivatized,protected monomer was coupled with a protected catechin monomer orprotected epicatechin monomer to form a protected 4,8-dimer which wasthen deprotected or used for further coupling with another protected,C-4 derivatized epicatechin monomer to form protected higher4,8-oligomers. If a 4,6-linkage was desired, the C-8 position of theprotected catechin or epicatechin monomer was blocked with a halogengroup prior to coupling with the C-4 derivatized, protected epicatechinmonomer or oligomer. Higher oligomers having both 4,8- and 4,6-linkageswere also prepared. The protected dimers or oligomers were deprotected,and if necessary, deblocked. The coupling was carried out in thepresence of a protic acid or a Lewis acid such as titanium tetrachloride(TiCl₄). The stereochemical nature of the interflavan bond was confirmedby the synthesis of a specifically protected derivative and itssubsequent degradation reference. Unfortunately, titaniumtetrachloride-mediated further chain extension of the epicatechin dimerleads to the formation of regioisomers. This is a serious drawback, notonly in terms of yield, but also purity. Even though the 4β,8-trimersand 4β,8-tetramers were isolated in pure form, the same cannotautomatically be expected for the larger oligomers, for which the numberof possible isomers, and thus contaminants, grows rapidly.

One potential way of dealing with this problem is to carefully purifythe chain-extended oligomer after each step in order to ensure that allchain-extended oligomers are at least derived from a single isomer ofthe starting oligomer. However, upon the titanium tetrachloride-mediatedchain extension of the protected trimer (2 eq.) with the C-4derivatized, protected monomer, not only were the protected tetramer,pentamer, and small amounts of higher oligomers formed, but theprotected trimer was degraded to the monomer and dimer, which thenparticipated in the chain-extension reaction, giving rise toregioisomeric oligomers such as small amounts of the protected4β,6:4β,8-trimer. While the reaction conditions (methylenechloride/tetrahydrofuran (9:11), 0° C., 15 min, then room temperature,140 min) were not optimized, the observation of this deleterious aidereaction warranted a search for a better synthetic approach.

Thus, there is a need for improved methods for synthesizing epicatechinor catechin oligomers, particularly the higher oligomers, and a processfor using protected larger epicatechin or catechin oligomers as buildingblocks for chain extension to even larger oligomers.

SUMMARY OF THE INVENTION

In one embodiment, bis(5,7,3′,4′-tetra-O-protected)-epicatechin and/or-catechin (4,8)-dimer and higher (4,8)-oligomers are prepared bycoupling a (5,7,3′,4′-tetra-O-protected)-epicatechin or -catechinmonomer with a 5,7,3′,4′-tetra-O-protected-4-(alkoxy)-epicatechin or-catechin monomer in the presence of an acidic clay. The protectinggroups used should not deactivate the A ring of the protected monomersor the A ring of the upper unit (i.e., mer) of the protected oligomers.The preferred protecting groups are benzyl groups. A suitable 4-alkoxygroup is a C₂–C₆ alkoxy group having a terminal hydroxy group,preferably 2-hydroxyethoxy. When the monomers are benzyl-protected, theprotected epicatechin (4β,8)-dimer is produced in significantlyincreased yields. Under the same conditions, the benzyl-protectedepicatechin (4,8)-trimer, -tetramer, and -pentamer are obtainedregioselectively from the next lower5,7,3′,4′-tetra-O-benzyl)epicatechin and/or catechin (4,8)-oligomer. Thepreferred acidic clay is a montmorillonite clay. The protected monomersand protected oligomers are separated by column chromatography, and thenthe protecting groups are replaced with hydrogen.

A process is also provided for chain extending atetra-O-benzyl-protected-epicatechin or -catechin monomers or oligomerswith a tetra-O-benzyl-protected-epicatechin or -catechin monomer havinga thiol C-4 activating group. The C-4 activated monomer is prepared byreacting a C-4 activated, tetra-O-protected-epicatechin or -catechinmonomer (e.g. 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechinwith a thiol derivatizing reagent such an organoaluminum thiolategenerated from 2-mercaptobenzothiazole or other heterocyclic thiol,e.g., 2-pyridinethiol, 4-pyridinethiol, or4-phenyl-1H-tetrazole-5-thiol. Preferably, to avoid the undesiredintervention of the C-3 hydroxyl group, this group is protected in boththe electrophilic and nucleophilic reaction partners by acetylation. Theacetylation is carried out after the tetra-O-benzyl protected monomer isactivated by introduction of the 4-thio group. The chain extension iscarried out in the presence of dimethy(methythio)sulfoniumtetrafluoroborate or preferably silver tetrafluoraborate. Preferably,the silver tetrafluoroborate is dried before the reaction. Morepreferably the drying is vacuum drying carried out immediately beforethe reaction. The resulting mixture comprises protected trimers throughprotected octamers. The protected oligomers are isolated by reversephase high pressure liquid chromatography. If present, the acetylprotecting group(s) are removed, preferably with aqueous tetra-n-butylammonium hydroxide. The benzyl protecting groups are removed byhydrogenolysis, preferably after removal of the acetyl protectinggroup(s) if such groups are present. With epicatechin, the yields arenear-quantitative. The oligomers are characterized as their peracetates.

In another embodiment, chain extension by cross-coupling of two5,7,3′,4′-tetra-O-benzyl-protected-epicatechin or -catechin(4β,8)-oligomers each having a C-4 thio activating group (e.g.,C-4-(2-benzlothiazolyl)thio) is carried out in the presence of silvertetrafluoroborate.

An improved process is also provided for preparing a5,7,3′,4′-tetra-O-benzyl-4-alkoxy-8-bromo-epicatechin or -catechinmonomer. In the prior art four step process for preparing the C-8blocked, C-4 alkoxy monomers the yield was about 51%. The steps included(i) the C-4 activation of 5,7,3′,4′-tetra-O-benzyl-epicatechin byreaction with ethylene glycol in the presence of2,3-dichloro-5,6-dicyano-1,4-benzoquinone(DDQ) (63% yield), (ii)protection of the 3-hydroxyl position of theC-4-(2-hydroxyethoxy)-5,7,3′,4′-tetra-O-benzylepicatechin by theintroduction of a tert-butyldimethylsilyl (TBDMS) group by reaction withtert-butyldimethylsilyl chloride in imidazole in the presence of4-dimethyl-aminopyridine (DMAP) (88% yield), (iii) introduction of an8-bromo group by reaction with N-bromosuccinimide at −40° C. inmethylene chloride (100%), and (iv) removal of the TBDMS group byreaction with tetrabutyl ammonium fluoride (TBAF) in tetrahydrofuran(92%). In the improved two step processes5,7,3′,4′-tetra-O-benzyl-epicatechin or -catechin is (i) activated byreaction with ethylene glycol in the presence of DDQ and (ii) blocked bybromination with N-bromosuccinimide or (i)5,7,3′,4′-(tetra-O-benzyl)-epicatechin or -catechin is blocked bybromination and then (ii) activated. The overall yield for the two stepprocess is about 63% when the monomer is epicatechin and the activationis done first and about 67% when the monomer is epicatechin and theblocking is done first.

The synthetic procyanidin oligomers are identical to the procyanidinoligomers isolated from cocoa bean extracts by normal-phase HPLC. Theregio- and stereochemistry of the interflavan linkages has beenestablished by partial thiolysis (see Hör, M. et al., Phytochemistry1996, 42, 109). For the tetramer the upper interflavan linkage is 4β,8and the lower portion of the molecule is identical to the trimer whichhas also been subjected to partial thiolysis with both linkages beingidentified as 4β,8 (see Hör et al.). Since in the course of the presentchain extension process the first three interflavan linkages formed areexclusively 4β,8 linkages, the same must be true for the additionalinterflavan linkages present in the higher oligomers.

When tested in several breast cancer cell lines, both the synthetic andnatural procyanidin pentamer, and to a lesser extent the tetramer,inhibited cell growth. Using the MDA MB-231 cell line, it wasestablished that this outcome is based on the induction of cell cyclearrest in the G₀/G₁ phase. Subsequent cell death is more likely necroticrather than apoptotic. Control experiments demonstrate that theprocyanidin itself, rather than hydrogen peroxide, is the causativeagent.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Chain Extension of Protected Eipicatechin Dimers and Trimers Mediatedby Acidic Clay

Chain extension of 5,7,3′,4′-tetra-O-benzylepicatechin and5,7,3′,4′-tetra-O-benzyl catechin mediated by an acidic clay such asMontmorillonite clay (e.g., Bentonite K-10) results in the almostexclusive formation of the protected (4β,8)-dimers. When the protectedmonomer was the epicatechin, the isolated yield was 90% together withsmall amounts of the protected (4β,8)₂-trimer, and no 4,6-linkedoligomers are observed. The surprisingly high reactivity differentialunder these conditions between the monomer and the dimer allows most ofthe dimer to survive without entering into further chain extension.Reaction of bis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer with5,7,3′,4′-tetra-O-benzylepicatechin activated at the C-4 position with a2-hydroxyethoxy group yields 40% of the (4β,8)₂-trimer together with 13%of the (4β,8)₃-tetramer by this chain extension protocol. The cleannessof this reaction permits, for the first time, at least a partialseparation of the monomer from the dimer, and even of the dimer from thetrimer, by column chromatography. This significantly reduces the amountof material that needs to be put through HPLC purification.

B. Chain Extension of 5,7,3′,4′-Protected or 3,5,7,3′,4′-ProtectedEpicatechin or Catechin Monomers Having A C-4 Thio Group

In an alternative chain extension, a 5,7,3′,4′-protected-epicatechin or-catechin is activated at the C-4 position with a thiol group such as2-(benzothiazolyl)thio. The chain extension is mediated by silvertetrafluoroborate. The reagent used to introduce the2-(benzothiazolyl)thio group at the C-4 position of an epicatechin or acatechin monomer is 2-mercaptobenzothiazole which is a non-volatile,odorless heterocyclic thiol. For this chain extension, C-4derivatized-C-5,7,3′,4′-benzyl-protected monomers, rather than theunprotected monomers, are preferred because they are easier to handle,more stable, and more accessible than the unprotected, C-4 derivatizedmonomers. The thio group can also be introduced by reaction with2-pyridinethiol, 4-pyridinethiol, or 1-phenyl-1H-tetrazole-5-thiol.

The monomer having the 2-(benzothiazoyl)thio activating group isprepared by reacting a protected epicatechin or catechin having a2-hydroxyethoxy group at the C-4 position with an organoaluminumthiolate prepared in situ from 2-mercaptobenzothiazole andtrimethylaluminum. See Dzhemilev U. M. et al., Izv. Akad. Nauk SSSR,Ser. Khim., 1988, 2645. The resulting 4-thioether is a mixture of twostereoisomers which are isolated by fractional crystallization andcolumn chromatography. Only the major stereoisomer is used for thesubsequent coupling but the minor stereoisomer can be used as well.

The chain extension is effected by adding silver tetrafluoride borate(AgBF₄) to a solution of 5,7,3′,4′-tetra-O-benzylepicatechin or5,7,3′,4′-tetra-O-benzylcatechin and the major stereoisomer. Thisresults in the formation of a protected procyanidin (4β,8)-dimer and(4β,8)₂-trimer. After normal-phase HPLC separation, the protected dimer,trimer, and monomer are recovered. Further separation by reverse-phaseHPLC yields, as a by-product, a protected 3-O-4 dimer. To avoid theundesired reaction of the 3-hydroxyl group in this chain extensionprocess, the 3-hydroxyl group is protected by acetylation of both thebenzyl-protected monomer and the benzyl-protected dimer. The yields arenear-quantitative. When a solution of silver tetrafluoroborate is addedto a solution of the acetyl- and benzyl-protected dimer and acetyl- andbenzyl-protected monomer, the expected acetyl- and benzyl-protectedtrimer and tetramer are formed, but only in low yields. The reason thatthe chain extension proceeds so slowly is that adventitious watersuccessfully competes with the flavanoid nucleophile, with the majorproduct of the coupling being the 4-hydroxy monomer3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-acetyl-4-hydroxyepicatechin. Inaddition, small amounts of the 4-hydroxy dimer,3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4β,8-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin),are also isolated, indicating self-condensation of the thioetherfollowed by either chain extension or hydrolysis.

In an attempt to improve the yield, the protected dimer and protectedmonomer are dried by stirring with powdered molecular sieves prior tothe addition of the silver tetrafluoroborate. The yield, however,remained unchanged. If the AgBF₄ is initially dried with molecularsieves, and the remaining reactants are added, no reaction takes place.Vacuum drying the silver tetrafluoroborate immediately before thecoupling solves the problem by reducing the hydrolysis of3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzlepicatechinto an acceptable level.

Using dry silver tetrafluoroborate with a protected monomer to protecteddimer molar ratio of 1:2.5, a series of protected oligomers spanningfrom the trimer, tris(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₂-trimer to the octameroctakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₇-octamer,can be isolated in a combined yield of 91%. The reaction isexceptionally clean, and no 4,6-oligomers are observed.

Similar results are obtained in the coupling of the C-4 derivatized,benzyl- and acetyl-protected monomer with the benzyl- andacetyl-protected trimer and tetramer. From these reactions, oligomers upto the protected undecamer can be isolated by reverse-phase HPLC ifethyl acetate (a nonpolar solvent) is admixed with the acetonitrile inthe final step of the gradient. The use of the ethyl acetate permitsrecovery of the highly retained higher oligomers; however, it alsoelutes significant amounts of aliphatic impurities which subsequentlyhave to be removed by additional HPLC steps, thus reducing the totalproduct recovery.

All of the protected oligomers (i.e., the benzyl ether-acetates) up tothe nonamer were deacetylated in near-quantitative yield with 40%aqueous tetra-n-butylanunonium hydroxide in tetrahydrofuran. This baseis used because of its good solubility in the relatively nonpolarreaction medium that is required by the lipophilicity of the startingmaterials. The ¹H NMR spectra of the resulting benzyl-ethers displayedsignals of two major rotamers together with trace amounts of additionalrotamers that increase as the oligomeric chain grows. It is believedthat these minor components are rotamers rather than regioisomersbecause similar signals are absent from the spectra of the precursoracetates. Samples of the benzyl ethers prepared in CDCl₃ exhibitwell-resolved, characteristic signals for the hydroxyl (OH) protons inthe δ 1.8–1.1 region.

The benzyl ethers (trimer through the octamers) are deprotected byhydrogenolysis over Pearlman's catalyst to form the unprotectedoligomers. Preferably, this deprotection is carried out inbicarbonate-washed glassware, as partial fragmentation to loweroligomers is occasionally observed without this precaution, quiteprobably as a consequence of the acidity of the glass surface of thereaction flask. To obtain a readily soluble procyanidin, it isnecessary, as similarly reported by others, to dilute the filteredsolution of the crude product with water, evaporate only partially so asto remove most of the organic solvents, and lyophilize the residualsolution. If the crude solutions are directly evaporated to dryness,partially insoluble materials result, indicating that some decompositionhas occurred. Combustion analyses shows that the lyophilized productscontain 1.3–2 equivalents of water per epicatechin moiety.

Comparison by normal phase HPLC analysis of epicatechin (4β,8)₂-trimer,epicatechin (4β,8)₃-tetramer, and epicatechin (4β,8)₄-pentamer was madeagainst the natural trimer, tetramer, and pentamer purified fromTheobroma cacao. Purities ranging from 94% to 96% were observed for thesynthetic procyanidins which were 2–4% higher than those for thenaturally-derived oligomeric procyanidins. The t_(R)'s of the syntheticprocyanidins match those observed for the natural oligomers, thusconfirming the epicatechin 4β,8 regio- and stereochemistry in thenatural cocoa procyanidins. All of the natural procyanidins purifiedfrom cocoa show impurity peaks preceding and following the main peak,with the tetramer and pentamer showing more impurity peaks. Scanningthese regions by HPLC/MS reveals no change in the [M]⁺ or [M+Na]⁺ ionsindicating that these minor impurities are isomers of the majoroligomers. These minor impurities may contribute to in vitro and in vivoactivities reported in the literature and potentially confoundstructure-activity relationships based only on natural oligomers. Hence,as a precaution, both natural and synthetic procyanidins are thereforeused in the biological assays reported in the following examples.

The nature of the impurities and of the side reaction(s) leading to themhas not been established but several trace impurities are present ratherthan a single major one. This is less than ideal; however, comparison ofreported optical rotations, for example, of the free (4β,8)-dimer or thetetramer reveals large variations that cannot merely be the consequenceof differential degrees of hydration, but appear to indicate thepresence of unknown impurities in some of these samples as well.

Since free polyphenols are inherently poorly amenable to purificationbecause of their oxygen and acid sensitivity (acid being required as asolvent additive to reduce peak tailing during HPLC), and their NMRspectra are anyway uncharacteristic because of severe line broadening,these compounds are characterized as their peracetates. The ¹H NMRspectra of the peracetates exhibit sharp signals for two rotamers (in a2;1 ratio for the trimer and in a 3:2 ratio for all higher homologs) andare, up to the heptamer or octamer, quite suitable for compoundidentification, since the acetate region serves as a useful“fingerprint”. As the oligomeric chain grows, the chemical shiftdifferences between analogous protons of epicatechin units in the innerposition of the chain become eventually insufficient at 300 MHz,resulting in the growth of uncharacteristic signal clusters without theappearance of well-separated new signals. These spectra can be usefulfor future reference. 13C NMR spectra are available for oligomers up tothe hexamer, beyond which insufficient amounts of material areavailable. The ¹H NMR spectra are of the trimer and tetramer in goodagreement with published spectra. See Hör, M. et al., Phytochemistry,1996, 42, for the trimer and tetramer and Sticher, O. F., Chromatogr. A,1999, 835, 59 for the trimer. In the case of the tetramer, partialthiolysis (see Hör et al.) establishes (4β,8)-regio- and-stereochemistry for the “upper” interflavan linkage, whereas the“lower” portion of the molecule is identical with the trimer. Thiscompound, in turn, has also been subjected to partial thiolysis, andboth interflavan linkages have been identified as (4β,8) (see Hör etal.). Since, therefore, in the chain extension process the first threeinterflavan linkages formed are exclusively of the 4β,8 type, the samemust be true for the additional interflavan linkages present in thelarger oligomers.

C. Cross Coupling of 3,5,7,3′,4′-Protected Monomers Having C-4(2-Benzothiazolyl)thio Groups and of a 3,5,7,3′,4′Protected OligomerHaving a C-4 (2-Benzothiazolyl)thio and a 3,5,7,3′,4′-Protected Oligomer

5,7,3′,4′-Tetra-O-benzylepicatechin monomers having2-(benzothiazolyl)thio groups at the C-4 position self-condensed in thepresence of silver tetrafluoraborate to yield a fairly complex mixturefrom which small amounts of the benzyl-protected,4-[(2-benzothiazolyl)thio]-dimer, -trimer, and presumably-tetramer canbe isolated. Also isolated are the rearranged benzyl-protected monomerand dimer where the group at the C-4 position is connected to thenitrogen rather than the sulfur of the thiazoyl ring. The migration ofthis moiety from sulfur to nitrogen is confirmed for the monomer by theobservation of a ¹³C NMR signal at δ 190.3 assignable to thethiocarbonyl carbon atom. The complexity of the above reaction mixtureis in part due to the formation of 4-O-3-linked oligomers similar to5,7,3′,4′-tetra-O-benzyl epicatechin4-O-3-(5,7,3,4-tetra-O-benzylepicatechin. AgBF₄-inducedself-condensation of3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinresults in low yields of the 4-[(2-benzothiazolyl)thio]-substitutedoligomers, i.e., dimer, trimer and tetramer. Together with theseproducts, and in considerable quantities because of the small reactionscale, the 4-hydroxy by-products are also formed. The by-products are3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin,3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin,and 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-O-benzyl-4-hydroxyepicatechin).

Reaction of[3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4-(2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]with tetrakis (3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin(4β,8)₃-tetramer in the present of silver tetrafluoroborate resulted inthe formation of the expected hexamer, i.e., hexakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin (4β,8)₅-hexamer in 12%yield together with the by-products octakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin 4β,8)₇-octamer,3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-4β,8-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin),and 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin4β,8-(3-O-acetyl-5,7,3′,4′-tetra-O-benzlepicatechin)-4β,8(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin).

Thus, chain elongation can be performed in increments of two flavanolunits. This procedure should be of use for the chain-extension of thelarger protected epicatechin oligomers as these compounds far exceed themonomeric 5,7,3′,4′-protected, C-4 derivatized building blocks in value.

D. Improved Process for Preparing 5,7,3′,4′-tetra-O-benzyl-epicatechinand -catechin Monomers having a Bromo Blocking Group at the C-8 Position

The C-4 position of a 5,7,3′,4′-tetra-O-benzyl-epicatechin or -catechinmonomer is activated by the introduction of an alkoxy group (e.g.,2-hydroxyethoxy) and the C-8 position is blocked with a bromo group. Theactivating and blocking step can be carried out in any sequence with theyield varying from about 63% to 67%. The activation is carried byreacting the protected monomer or the blocked protected monomer.Ethylene glycol in the presence of DDQ. The protection is carried out byreacting the protected monomer or activated, protected monomer withN-bromosuccinimide at −40° C. in methylene chloride.

E. Preparation of Procyanidin (4,8)-Dimer Digallates

Epicatechin- and/or catechin-(4,8) dimer digallates are prepared byesterifying octa-O-protected (4,8) dimers with hydroxy-protected gallicacid, preferably bydroxy-galloyl acid chloride. The preferred protectinggroups for both the dimer and the acid or acid halide are benzyl groups.The dimer digallates which can be prepared includeepicatechin-(4β,8)-epicatechin digallate, epicatechin-(4β,8)-catechin-(4α,8)-catechin digallate, catechin-(4β,8)-catechin digallate,catechin-(4α,8)-epicatechin digallate, and catechin-(4β,8)-epicatechindigallate.

A tetra-O-protected (4,8)-epicatechin and/or -catechin dimer (e.g.,5,7,3′,4′-tetra-O-benzylepicatechin) is esterified with tri-O-benzylgalloyl halide. Tri-O-benzyl gallic acid can be converted in situ into ahalide, e.g., chloride. The esterification is carried out in pyridinesolution in the presence of 4-dimethylaminopyridine (DMAP).Hydrogenolysis over 20% Pd (OH)₂/C gives the deprotected bisgallate.When the dimer is an epicatechin-(4β,8)-epicatechin, the deprotectedbisgallate is recovered in 90% yield as a hydrate without need forchromatographic purification (5.3 equiv. of water per bisgallatemolecule).

The preparation of epicatechin-(4β,8)-epicatechin bisgallate isdisclosed in “Studies in Polyphenol Chemistry and Bioactivity. 1.Preparation of Building Blocks from (+)-catechin. Procyanidin Formation.Synthesis of the Cancer Cell Growth Inhibitor,3-O-Galloyl-(2R,3R)-epicatechin-(4β,8)-[3-O-galloyl-(2R,3R)-epicatechin]by W. Tückmantel et al., J. Am. Chem. Soc., 1999, 121, 12073–12081. Thepreparation of gallic acid esters of epicatechin-(4α,8)-epicatechinbisgallate is disclosed in “Studies in Polyphenol Chemistry and Biology.3. Stereocontrolled Synthesis of Epicatechin-4α,8-epicatechin, anUnnatural Isomer of the β-Type Procyanidin, Alan P. Kozikowski et al.,J. Org. Chem., 2001, 66, 1287–1295.

Reagents, Test Procedures, and Analytical Procedures Reagents

Pearlman's catalyst (20% Pd(OH)₂/C) was obtained from Aldrich andcontained up to 50% H₂O. Bentonite K-10 was purchased from Acros. Forother chemicals, see Tückmantel, W. et al., J. Am. Chem. Soc., 1999,121, 12073.

Acetylation

Since the free procyanidins are poorly amenable to purification becauseof their oxygen and acid sensitivity and their NMR spectra areuncharacteristic because of severe line broadening, these compounds arecharacterized as their peracetates. The H¹ NMR spectra of theperacetates exhibit sharp signals for two rotameters (in a 2:1 ratio forthe trimer and in a 3:2 ratio for all higher oligomers). The spectra upto the octamer are quite suitable for compound identification. Theacetate region serves as a useful “fingerprint”. 13C NMR spectra havebeen acquired for oligomers up to the hexamer.

The ¹H NMR spectra for the trimer and tetramer are in good agreementwith those published.

Spectra

¹H and ¹³C NMR spectra were acquired at nominal frequencies of 300 and75 MHz, respectively, in CDCl₃ unless specified otherwise. ¹H NMRspectra are referenced to internal TMS; ¹³C NMR spectra to internal TMSif so marked or otherwise to the CDCl₃ signal (δ 77.00). Combustionanalyses were carried out by Micro-Analysis, Inc. (Wilmington, Del.).

Column Chromatography

Column chromatography (CC) was carried out on Merck silica gel 60 (No.7734-7), particle size 63–200 μm. TLC: Merck silica gel 60 F₂₅₄ (No.7734-7), layer thickness 250 μm; visualization by UV light or withalkaline KMnO₄ solution.

High Pressure Liquid Chromatopraphic (HPLC) Analysis of Procyanidins

Chromatographic analyses of free procyanidin oligomers were performed ona HP 1100 HPLC system (Hewlett Packard, Palo Alto, Calif.) equipped withan autoinjector, quaternary HPLC pump, column heater, diode arraydetector, fluorescence detector, and HP ChemStation for data collectionand sample manipulation. Normal phase separations were performed on a250×4.6 mm Phenomenex (Torrance, Calif.) 5 μm Prodigy column. Thedetector was a fluorescence detector operating at λ_(ex)=276 nm andλ_(em)=316 nm. The ternary mobile phase consisted of (A)dichloromethane, (B) methanol and (C) acetic acid:water (1:1 v/v).Separations were effected by a series of linear gradients of B into Awith a constant 4% C at a flow rate of 1 mL/minutes as follows: 0–30minutes, 14.0–28.4% B in A; 30–50 minutes, 28.4–38.0% B in A; 50–51minutes, 38.0–86.0% B in A; 51–56 minutes, 86.0% B in A isocratic.

Other HPLC: columns: column A, Hewlett-Packard RP-8, 200×4.6 mm, at 1.0mL/min; column B, Waters μBondapak C₁₈, 300×7.8 mm, at 2.8 mL/min;column C, Waters μBondapak C₁₈, 300×19 mm; column D, Waters μBondapakC₁₈, 300×30 mm, at 42 mL/min; column E, Whatman Partisil 10, 500×9.4 mm,at 5.0 mL/min; column F, Whatman Partisil 10, 500×22 mm, at 26 mL/min.Detection was by UV absorption at 280 nm. Retention times variedsubstantially depending on column history and other subtlecircumstances. They are quoted solely for orientation and should not beemployed for product identification without comparison to an authenticreference sample. See examples for further details.

High Pressure Liquid Chromatgraphic/Mass Spectra (HPLC/MS) Analysis ofProcyanidins

HPLC/MS analyses of natural and synthetic procyanidins were performed onan HPLC system (as described above) which was interfaced to an HP Series1100 mass selective detector (Model G1946A) equipped with an API-ESionization chamber. Ionization reagents were added via a tee in theeluent stream just prior to the mass spectrometer. Conditions foranalysis in the positive ion mode included the introduction of 0.05 Msodium chloride at a flow rate of 0.05 mL/minutes to assist ionization,a capillary voltage of 3.5 kV, a fragmentor voltage of 100 V, anebulizing pressure of 25 psig, and a drying gas temperature of 350° C.Scans were performed over a mass range of m/z 100–3000 at 1.96 s percycle.

Cell Lines

The human breast cancer cell lines MCF-7, SKBR-3, MDA 435, and MDAMB-231 were obtained from the Lombardi Cancer Center Cell Culture CoreFacility at Georgetown University Medical Center. The MDA MB-231 cellline was P53 defective, ER negative, and constitutively expressed K-ras.Cells were cultured in T-75s in IMEM medium (BioFluids Inc.)supplemented with 10% FBS (Gibco BRL Life Technologies) in a humidified5% CO₂ atmosphere at 37° C.

Cytotoxicity Assay

Cytotoxicity assays were performed on several human breast cancer celllines treated with test compounds in a 96 well microtiter plate formatusing the microculture tetrazolium assay²⁸ modified for use with crystalviolet rather than MTT. Briefly, 1–2×10³ cells were added per well andallowed to culture in a humidified, 5% CO₂ atmosphere until they reachedapproximately 50% confluence. Sterile filtered test compounds were addedat various concentrations, and the plates were allowed to culture for anadditional 12–36 hours. The growth medium was then removed, and eachwell was washed twice with 200 μL each of pH 7.4 PBS. After washing, 50μL of filtered crystal violet solution (2.5 g/125 mL of methanol+375 mLof H₂O) was added. At the end of 5 minutes, the crystal violet wasremoved, and the plate was washed three times with water. Plates wereallowed to dry, and the crystal violet stained cells were resolubilizedin 100 μL of 0.1 M sodium citrate in ethanol/water (1:1, v/v). At theend of 1 hour, the plates were scanned at 570 nm (ref. 405 nm) with aMolecular Devices Corporation microtiter plate reader, and the data wasrecorded with the SOFTMAX software program. The average of 3 readingswas taken for each blank, control, vehicle, and test concentration forstatistical data manipulation.

Flow Cytometry

MDA MB-231 cells were cultured as described above until they reachapproximately 50% confluence. Sterile filtered test compounds orcatalase (Sigma #C9322) adjusted to 100 U/mL or heat inactivatedcatalase (solution immersed in boiling water for 15 min) or hydrogenperoxide (H₂O₂) was then added, and the cells were allowed to culturefor an additional 24 h. The cells were then trypsinized and counted, and1.5×10⁶ cells were taken for cell cycle analysis by the Vindelov method.See Vindelov, L. et al., “A Detergent Trypsin Method for the Preparationof Nuclei for Flow Cytometric DNA Analysis”, Cytometry, 1983, 3,323–327. Analyses were performed by the Lombardi Cancer Center FlowCytometry Core Facility at Georgetown University Medical Center.

Annexin V-FITC

The annexin V-FITC assay was performed on procyanidin-treated MDA MB-231cells using the TACS™ Annexin V-FITC kit (Trevigen Inc.) according tothe manufacturer's procedure.

EXAMPLES Example 1 Preparation of Tetra-O-Protected, C-4ActivatedMonomers

Part A—Preparation of5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin

To a solution of 21.5 g (33.0 mmol) of5,7,3′,4′-tetra-O-benzylepicatechin in 220 mL of anhydrous methylenechloride (CH₂Cl₂) was added at room temperature 11.0 mL (198 mmol) ofethylene glycol and then all at once with good stirring 15.0 g (66 mmol)of 2,3-dichloro-5,6-dicycano-1,4-benzoquinone(DDQ) was added. After 110minutes of vigorous stirring at room temperature under a calciumchloride (CaCl₂) tube, a solution of 8.5 g (69.5 mmol) of4-(dimethylamino)pyridine (DMAP) in 50 mL of anhydrous methylenechloride was added whereupon a copious dark precipitate appeared. Afteranother 10 minutes of stirring at room temperature, the mixture wasfiltered over a coarse glass frit, the precipitate was washed with 50 mLof methylene chloride, and the solution was evaporated to near dryness.The residue was filtered over silica gel (17×9 cm) with ethylacetate/hexane 1:1, and all product-containing fractions were pooled.After evaporation to approximately 75 mL, crystals began to appear(seeding may be necessary). An equal volume of hexane was added, andcrystallization was allowed to proceed at room temperature overnight.Suction filtration, washing twice with 25 mL of ethyl acetate/hexane(1:2), and drying in vacuo (initially at room temperature, then at 40°C.) furnished 10.6 g (45%) of5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin as an off-whitesolid. It was analyzed by HPLC and found to be 97% pure AKA acetanitrile(column B; 0–20 minutes 50 to 100% methylcyanide (CH₃CN) in H₂O, thenCH₃CN; t_(R) 17.9 min). Additional product was obtained from the motherliquor by column chromatography on silica (SiO₂) (33×5 cm) and elutionwith ethyl acetate/hexane (1:2) (forerun), then 2:3 (product). Afterevaporation, the resulting amber glass (1.0 g, purity 69%) wascrystallized twice from ethyl acetate/hexane to yield another 0.5 g (2%)of 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin (purity 98%).

Part B—Preparation of5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)catechin

Using the procedure described above the5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)catechin was prepared.

Example 2 Condensation of Tetra-O-Protected Catechin or EpicatechinMonomers with Tetra-O-Protected, C-4 Activated Catechin or EpicatechinMonomers Catalyzed by Acidic Clay

Part A—Condensation of 5,7,3′,4′-Tetra-O-benzylepicatechin with5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin

To a solution/suspension of 9.26 g (14.2 mmol, 4 equiv.) of5,7,3′,4′-tetra-O-benzyl-epicatechin and 5.0 g of Bentonite K-10 clay in115 mL of anhydrous methylene chloride (CH₂Cl₂) was added, with icecooling, stirring and exclusion of moisture, within 2.5 hours 2.53 g(3.56 mmol) of 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechinin 35 mL of anhydrous methylene chloride. The bath temperature rose to+6° C. at the end of the addition. Stirring in the bath was continuedfor 1 hour, during which time the temperature rose to +12° C. The claywas filtered off with suction over celite, and the solids were washedtwo times with 50 mL of methylene chloride. Twenty mL of toluene wasadded, and the solution was evaporated to a small volume. The residuewas chromatographed on silica gel (60×5 cm) with ethylacetate/chloroform/hexane (1:14:14). Initially, 5.95 g of unreacted5,7,3′,4′-tetra-O-benzlepicatechin was eluted, followed by 4.01 g ofprotected monomer/protected dimer mixed fractions and 1.15 g of pure(98% by HPLC) bis(5,7,3′,4′-tetra-O-benzylepicatechin (4β,8)-dimer. Thelast traces of the dimer together with the protected trimer were elutedas a mixed fraction (0.27 g) with a solvent ratio of 1:7:7.

The mixed fractions were each dissolved in methyl cyanide (CH₃CN) andseparated by preparative HPLC (column D; 0–30 minutes, 80 to 100%(CH₃CN) in H₂O, then CH₃CN; the retention times for the protected dimerand protected trimer were 23.3 and 30.1 minutes, respectively. Aftercombination of the appropriate fractions, evaporation, and drying invacuo, the following yields were obtained:5,7,3′,4′-tetra-O-benzylepicatechin, 6.89 g (74% recovery);bis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer, 4.26 g (92%);tris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer 74 mg (2%).Bis-(5,7,3′,4′-tetra-O-benzyl)-epicatechin (4β,8)-dimer. ¹³C NMR (CDCl₃,TMS) δ 158.34, 158.07, 157.91, 157.07, 156.83, 156.56, 156.49, 155.89,155.53, 155.07, 154.44, 152.83, 149.17, 149.01, 148.92, 148.66, 148.60,148.40, 148.18, 137.40, 137.38, 137.30, 137.28, 137.22, 137.17, 137.01,136.97, 132.61, 132.43, 131.18, 131.14, 128.6–126.6, 119.96, 119.79,118.79, 118.65, 115.02, 114.89, 114.35, 114.05, 113.52, 112.93, 112.46,111.58, 111.17, 104.45, 102.29, 101.76, 94.34, 93.96, 93.33, 93.15,92.93, 91.52, 78.84, 78.07, 75.63, 72.41, 72.14, 71.48, 71.35, 71.22,70.81, 70.48, 69.92, 69.86, 69.78, 69.47, 69.05, 66.50, 65.15, 35.90,35.78, 28.74, 28.61. Other data have been published (see Part 1:Tückmantel, W. et al. J. Am. Chem. Soc., 1999, 121, 12073).

In another run (3.17 mmol of5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-epicatechin), anessentially complete separation of protected monomer and protected dimerand of protected dimer and protected trimer was achieved during columnchromatography, with only the protected trimer and the very dilute tailof the protected dimer requiring purification by HPLC. The followingyields were obtained: 5,7,3′,4′-tetra-O-benzylepicatechin, 6.20 g (75%recovery); bis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer, 3.63 g(88%); and tris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer,0.15 g (5%).

Part B—Condensation of 5,7,3′,4′-Tetra-O-benzylcatechin with5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin

Using the procedure described above, a protectedepicatechin-(4β,8)-protected catechin dimer was prepared. The purityranged from 89–98%.

Part C—Condensation of 5,7,3′,4′-Tetra-O-benzylcatechin with5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy) protected catechin

Using the procedure described above, a protectedcatechin-(4β,8)-protected catechin dimer was prepared. The purity rangedfrom 93–98%.

Part D—Condensation of 5,7,3′,4′-Tetra-O-benzylcatechin with5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy) protected catechin

Using the procedure described above, a protectedcatechin-(4α,8)-protected catechin dimer was prepared. The purity rangedfrom 97–98%.

Part E—Condensation of (5,7,3′,4′-tetra-O-benzyl)catechin with(5,7,3′,4′-tetra-O-benzyl)-4-(2-hydroxyethoxy)catechin

Using the procedure described above, a protectedcatechin-(4β,8)-protected epicatechin dimer can be prepared by reacting(5,7,3′,4′-tetra-O-benzyl)-epicatechin and (5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-catechin.

Part F—Condensation of (5,7,3′,4′-tetra-O-benzyl)catechin with5,7,3′,4′-(tetra-O-benzyl)-4-(2-hydroxyethoxy)epicatechin

Using the procedure described above, a protectedcatechin-(4α,8)-protected epicatechin dimer can be prepared by thecondensation of 5,7,3′,4′-tetra-O-benzyl-epicatechin with(5,7,3′,4′-tetra-O-benzyl)-4-(2-hydroxyethoxy)catechin.

Example 3 Condensation of Bis(5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)-Dimer with5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin Catalyzed byAcidic Clay

To a solution/suspension of 5.60 g (4.31 mmol, 3 equiv.) ofbis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer and 2.04 g ofBentonite K-10 clay in 45 mL of anhydrous methylene chloride (CH₂Cl₂)was added, with ice cooling, stirring and exclusion of moisture, within110 minutes 1.02 g (1.44 mmol) of5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-epicatechin in 15 mL ofanhydrous methylene chloride (CH₂Cl₂). The bath temperature rose to +6°C. at the end of the addition. Stirring in the bath was continued for 1hour, during which time the temperature rose to +12° C. The clay wasfiltered off with suction over celite, and the solids were washed fourtimes with 25 mL each of ethyl acetate. The combined solutions wereevaporated. Attempted separation by column chromatography on silica gel(56×5 cm) with ethyl acetate/hexane/chloroform (1:10:10) failed toseparate the dimer and the trimer. Subsequent elution with a solventratio of 1:7:7 gave 0.50 g of a fraction consisting mostly of tetramertogether with residual trimer. The dimer/trimer fraction was againsubjected to column chromatography on silica gel (55×5 cm), this timestarting with ethyl acetate/chloroform/hexane 1:14:14. After elutionwith 20 L of this mixture, the solvent ratio was switched to 1:12:12 (5L), then 1:10:10, resulting in the recovery of 4.40 g of the dimer.Further elution with a mixing ratio of 1:8:8 gave 1.04 g of crude trimer(purity 90% by HPLC).

The crude trimer and the trimer/tetramer mixture were each dissolved inmethyl cyanide (CH₃CN) and separated by preparative HPLC (column D; 0–30minutes, 80 to 100% CH₃CN in water, then CH₃CN); the retention times forthe dimer, trimer, and tetramer were 22.5 (22.7), 30.1 (30.8), and 33.9minutes, respectively. After combination of appropriate fractions,evaporation, and drying in vacuo, the following yields were obtained:bis(5,7,3′,4′-tetra-O-benzyl)epicatechin 4β,8)-dimer, 4.43 g (79%recovery); tris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer,1.13 g (40%); tetrakis-(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₃tetramer, 0.24 g (13%).

Example 4 Preparation of4-[(2-Benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin

To a solution of 6.5 g (39 mmol) of 2-mercaptobenzothiazole in 40 mL of1,2-dichloroethan [HPLC grade, filtered over basic alumina (activity I)immediately before use] was added dropwise in 10 minutes under nitrogenwith ice cooling and stirring 19.5 mL of trimethylaluminum solution (2.0M in toluene). The resulting amber solution was stirred at 0° C. for 15minutes, then a solution of 5.56 g (7.82 mmol) of5,7,3′,4′,-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin in 60 mL of1,2-dichloroethane (pretreated as above) was added dropwise in 20minutes. The orange-colored reaction mixture was stirred at roomtemperature for 5 hours, then cooled in an ice bath, and a solution of22.6 g (80 mmol) of potassium sodium tartrate tetrahydrate in 90 mL ofwater and 100 mL of 2.5 M aqueous sodium hydroxide was added dropwise(very cautiously at first because of gas evolution). Methylene chloride(100 mL) was added, and the phases were separated. The organic phase waswashed two times with 100 mL of 2.5 M aqueous sodium hydroxide and driedover sodium sulfate. After evaporation to a small volume, the residuewas chromatographed on a short silica gel column with ethylacetate/toluene 1:19 (until the beginning elution of product), then 1:9.The eluate was evaporated to yield an oil, which soon turned into alight-yellow solid. This material was dissolved in 30 mL of hot ethylacetate, 90 mL of 1-chlorobutane was added, and the solution was seededand set aside for crystallization first at room temperature, then at−20° C. The precipitate was isolated by suction filtration, washed twotimes with 20 mL of cold 1-chlorobutane, and dried in vacuo to yield3.50 g of the predominant diastereoisomer. Chromatography of the motherliquor (silica gel, ethyl acetate/methylene chloride/hexane 1:18:11 to2:18:11) followed by crystallization from ethyl acetate/1-chlorobutaneyielded an additional 0.78 g of the major isomer (together 4.28 g, 67%)and 0.16 g (2.5%) of the less polar minor isomer.

Major diastereoisomer: mp 160–161° C. (fromethylacetate/1-chlorobutane); [α]_(D)+106°, [α]₅₄₆+133° (EtOAc, c 10.6gL⁻¹); ¹H NMR (CDCl₃) δ 7.89 (ddd, 1 H, J=8, 1.2, 0.7 Hz), 7.78 (ddd, 1H, J=8, 1.2, 0.7 Hz), 7.47–7.20 (m, 19 H), 7.17 (d, 1 H, J=2 Hz),7.12–7.00 (m, 4 H), 6.95 (B part of an ABq, 1 H, J=8.5 Hz), 6.30, 6.29(ABq, 2 H, J=2 Hz), 5.46 (d, 1 H, J=2 Hz),5.42 (s, 1 H), 5.17 (s, 2 H),5.16 (s, 2 H),5.10, 5.05 (ABq, 2 H, J=12 Hz), 5.03 (s, 2 H), 4.40 (ddd,1 H, J=6, 2.5, 1 Hz), 2.00 (d, 1 H, J=5.5 Hz); ¹³C NMR (CDCl₃) δ 165.00,160.67, 158.76, 155.95, 153.16, 148.96, 148.88, 137.17, 137.07, 136.53,136.47, 135.29, 130.76, 128.61, 128.45, 128.37, 128.16, 128.09, 127.75,127.56, 127.49, 127.46, 127.21, 126.57, 126.06, 124.41, 121.83, 120.97,119.65, 114.91, 113.58, 98.34, 94.48, 75.13, 71.32, 71.22, 70.78, 70.13,69.86, 44.43; IR (film) 3554 (br), 1617, 1591, 1177, 1152, 1114, 735,696 cm⁻¹. Analysis Calcd for C₅₀H₄₁NO₆S₂: C, 73.60; H, 5.06; N,1.72.Found: C, 73.92; H, 4.75; N, 1.74.

Minor diastereoisomer: mp 144–146° C. (from ethylacetate/1-chlorobutane); [α]_(D)−48.9°, [α]₅₄₆−64.6° (EtOAc, c 7.6gL⁻¹); ¹H NMR (CDCl₃) δ 7.79 (ddd, 1 H, J=8, 1.2, 0.7 Hz), 7.66 (ddd, 1H, J=8, 1.2, 0.7 Hz), 7.47–7.25 (m, 14 H), 7.17–7.11 (m, 2 H), 7.08–6.89(m, 5 H), 6.84–6.77 (m, 4 H), 6.27, 6.25 (ABq, 2 H, J=2 Hz), 5.45–5.40(m, 2 H), 5.16 (narrow ABq, 2 H), 5.11, 5.07 (ABq, 2 H, J=13 Hz), 5.07,5.03 (ABq, 2 H, J=11.5 Hz), 4.94, 4.87 (ABq, 2 H, J=11.5Hz), 4.78 (q, 1H, J=5Hz), 4.39 (d, 1 H, J=5 Hz); ¹H NMR (C₆D₆) δ 7.68 (d, 1 H, J=8 Hz),7.38 (d, 1 H, J=2 Hz), 7.32–6.96 (m, 19 H), 6.90–6.68 (m, 6 H), 6.48 (d,1 H, J=2 Hz), 6.22 (d, 1 H, J=2.5 Hz), 5.82 (dd, 1 H,J=5, 1.2 Hz), 5.57(d, 1 H, J=4.5 Hz), 4.95 (s, 2 H), 4.82 (q, 1 H, J=4.5 Hz), 4.80 (s, 2H), 4.71 (s, 2 H), 4.70 (d, 1 H, partly concealed), 4.58, 4.51 (ABq, 2H, J=12 Hz); ¹³C NMR (CDCl₃) δ 169.70, 160.84, 158.13, 155.45, 152.30,148.53, 148.14, 137.14, 137.02, 136.41, 135.88, 135.44, 129.67, 128.58,128.36, 128.16, 128.08, 127.87, 127.65, 127.55, 127.35, 127.31, 127.22,127.00, 126.70, 125.89, 124.15, 121.23, 120.85, 119.74, 114.41, 114.31,100.87, 93.80, 93.76, 76.46, 71.01, 70.73, 70.06, 70.00, 68.02, 46.51;IR (film) 3440 (br), 1614, 1584, 1154, 1122, 752, 732, 696 cm⁻¹.Analysis: Calculated for C₅₀H₄₁NO₆S₂: C, 73.60; H, 5.06; N, 1.72. Found:C, 73.22; H, 4.64; N, 1.71.

The above reaction should be conducted in a well-ventilated fume hoodbecause although 2-mercaptobenzothiazole is odorless, small quantitiesof malodorous (but not very volatile) 2-(benzylthio) benzothiazole wasformed in this reaction.

Example 5 Preparation of3-O-Acetyl-4-[(2-benzothiazolyl)thiol]-5,7,3′,4′-tetra-O-benzylepicatechin

To a solution of 3.50 g (4.29 mmol) of4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin (majordiastereomer from Example 4) and 53 mg (0.43 mmol) of4-(dimethylamino)pyridine in 12 mL of anhydrous pyridine was added allat once 2.0 mL (21.5 mmol) of acetic anhydride. The reaction mixture waskept at room temperature in a closed flask for 50 hours. Ice and 150 mLof 5% aqueous hydrochloric acid were added. The product was extractedinto 100+20 mL of methylene chloride. The combined organic phases werewashed with 100 mL of water and two times with 50 mL of 10% aqueoussodium hydroxide; after each washing, the aqueous phase wasback-extracted with 20 mL of methylene chloride. The combined organicphases were dried over magnesium sulfate and evaporated and the residuewas taken up in a small volume of toluene and filtered over silica gelwith ethyl acetate/hexane (1:3). Evaporation and drying in vacuo yielded3.58 g (97%)3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinas a yellowish foam: [α]_(D)+91.7°, [α]₅₄₆+115° (EtOAc, c 13.2 gL⁻¹); ¹HNMR (CDCl₃) δ 7.90 (d, 1 H, J=8 Hz), 7.77 (d, 1 H, J=8 Hz), 7.46–7.22(m, 19 H), 7.11 (d, 1 H, J=2 Hz), 7.09–7.00 (m, 3 H), 6.99, 6.91 (ABq, 2H, J=8.5 Hz, A part d with J=2 Hz), 6.31, 6.30 (ABq, 2 H, J=2.5 Hz),5.63 (dd, 1 H, J=2.5, 1.2 Hz), 5.55 (s, 1 H), 5.31 (d, 1 H, J=2 Hz),5.17, 5.12 (ABq, 2 H,J=12 Hz), 5.14 (s, 2 H), 5.10, 5.05 (ABq, 2 H, Jnot readable because of overlap), 5.07, 5.02 (ABq, 2 H, J=11.5 Hz), 1.84(s, 3 H); ¹³C NMR (CDCl₃, TMS) δ 169.08, 164.07, 160.69, 158.31, 156.03,153.22, 148.92, 148.89, 137.18, 137.16, 136.53, 136.31, 135.62, 130.29,128.67, 128.45, 128.24, 128.19, 127.78, 127.65, 127.43, 127.31, 126.87,126.10, 124.50, 122.16, 121.01, 119.80, 114.97, 113.51, 98.50, 94.46,94.30, 74.13, 71.44, 71.23, 70.74, 70.19, 70.13, 42.59, 20.84; IR 1750,1616, 1591, 1217, 1152, 1117, 734, 696 cm⁻¹. Analysis: Calculated forC₅₂H₄₃NO₇S₂: C, 72.79; H, 5.05; N, 1.63. Found: C, 73.01; H, 4.79; N,1.61.

Example 6 Preparation ofBis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-Dimer

A solution of 1.5 mL (16 mmol) of acetic anhydride in 4 mL of anhydrouspyridine was added all at once to 3.69 g (2.84 mmol) ofbis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer. The mixture wasoccasionally swirled until all starting material dissolved and thenallowed to stand in a closed flask at room temperature for 99 hours. Thereaction was terminated by addition of 30 mL of ethyl acetate and 2 mLof methanol and allowed to stand at room temperature for 1.5 hours.Another 20 mL of ethyl acetate was added. Then the solution was washedwith 200 mL of 0.5 M aqueous phosphoric acid (H₃PO₄). The aqueous layerwas back-extracted with 50 mL of ethyl acetate. The combined organicphases were dried over magnesium sulfate. After evaporation, the residuewas taken up in a small volume of toluene and chromatographed on a shortsilica gel column with ethyl acetate/hexane (1:9, then 1:3, finally1:1). Evaporation and drying in vacuo yielded 3.82 g (97%) ofbis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer as acolorless foam.

Example 7 Reaction of3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinwith Bis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-Dimer

A 0.80 g (4.1 mmol) sample of silver tetrafluoroborate (AgBF₄) was driedin the reaction flask at 100° C. in an oil pump vacuum with exclusion oflight for 1.5 hours. After cooling, the vacuum was broken with nitrogen,and a solution of 5.66 g (4.09 mmol) ofbis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer in 60mL of anhydrous tetrahydrofuran was added all at once. The flask wasplaced in an ice bath under dim light, and a solution of 1.40 g (1.64mmol) of3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzyleipcatechinin 30 mL of anhydrous tetrahydrofuran was added dropwise in 70 minuteswith stirring. The reaction mixture turned yellow, and a turbidityeventually appeared. Stirring at 0° C. was continued for 40 minutes,during which time period the reaction mixture turned into a milky,whitish suspension. Triethylamine (1.1 mL, 8 mmol) was added, themixture was evaporated to near dryness, and the residue was filteredover a short silica gel column with ethyl acetate/hexane 1:1. The eluatewas evaporated and the crude product was analyzed by HPLC (column A;0–30 minutes, 80 to 100% methyl cyanide (CH₃CN) in water, then CH₃CN.The following peaks were observed (assignment/area %): t_(R) 5.0(4-OH-monomer, 0.15), 12.6 (4-OH-dimer, 0.25), 15.6 (dimer, 59.4), 24.8(trimer, 23.4), 30.3 (tetramer, 12.5), 33.3 (pentamer, 3.2), 35.4(hexamer, 0.8), 37.3 (heptamer, 0.1), 39.1 minutes (octamer, 0.02). Apartial separation was achieved by column chromatography on silica gel(38×9 cm). Initial elution with 25 L of ethyl acetate/chloroform/hexane(1:10:9) did not result in product recovery (this stage was, however,essential for achieving separation). Another 25 L of ethylacetate/chloroform/hexane (1:11:8) eluted 4.01 g of the dimer (71%recovery; pure by HPLC). A fraction (1.72 g) consisting of trimer,tetramer, and some pentamer was eluted with 20 L of ethylacetate/chloroform/hexane 1:12:7. Finally, the column was stripped withethyl acetate/chloroforn/hexane (2:12:7) to give 0.87 g of a fractionconsisting mostly of the larger oligomers. The latter two fractions weretaken up in methyl cyanide (CH₃CN) and separated in several portions bypreparative HPLC (column D; 0–30 minutes, 80 to 100% CH₃CN in water,then CH₃CN, and the appropriate fractions were pooled and dried in vacuoto obtain the oligomers as colorless films or foams. The retention timesand yields relative to3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinfor the trimer through octamer were 31.9 minutes (1.46 g, 43%), 36.0minutes (755 mg, 33%), 39.6 minutes (204 mg, 11%), 45.0 minutes (45 mg,2.6%), 52.8 minutes (13.8 mg, 0.9%), and 64.1 minutes (5.2 mg, 0.3%),respectively; for3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)22.2 minutes (13.4 mg, 1.2%). The 4-OH monomer (i.e.,3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin) was notrecovered from the silica gel column, probably because of its highpolarity. The total mass balance relative to3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinwas 92%.

Example 8 Coupling of3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinwith Tris-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₂-Trimer

The reaction was conducted analogously to the coupling of Example 7using 0.41 g (2.1 mmol) of silver tetrafluoroborate (AgBF₄), 4.40 g(2.12 mmol) of tris(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin(4β,8)₂-trimer, and 729 mg (850 μmol) of3-O-acetyl-4-[2-(benzoyhiazolythio]-5,7,3′,4′-tetra-O-benzylepicatechin.After filtration over silica gel with ethyl acetate/hexane (1:1), thecrude product was taken up in methyl cyanide (CH₃CN) and separated inseveral portions by preparative HPLC as above to yield the followingproducts: 3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin (31mg, 5%);3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin(34 mg, 6%); trimer (3.07 g, 70% recovery); tetramer (1.47 g, 62%);pentamer (221 mg, 15%); hexamer (57 mg, 5%); heptamer (25.2 mg, 2%);octamer (10.8 mg, 1%). Total mass balance relative to3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin96%.

Example 9 Coupling of3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinwith Tetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₃-Tetramer

The reaction was conducted analogously to the couplings of Examples 7and 8 using 0.34 g (1.75 mmol) of silver tetrafluoroborate (AgBF₄), 4.77g (1.73 mmol) oftetrakis-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₃-tetramer, and 592 mg (690 μmol) of3-O-acetyl)-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin.After filtration over silica gel with ethyl acetate/hexane (1:1), thecrude product was subjected in several portions to a preliminaryseparation by preparative HPLC (column D; 0–30 minutes, 80 to 100%methyl cyanide (CH₃CN) in water; 30–38 minutes, CH₃CN; 38–65 minutes,10% ethyl acetyl in CH₃CN) to yield the following products:3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxy epicatechin (t_(R) 11.0min; 19 mg, 4%);3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)₃-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin(22.2 min; 47 mg, 10%); tetramer (36.0 min; 3.56 g, 74% recovery);pentamer (39.4 min; 1.03 g); hexamer (43.6 min; 260 mg); heptamer (46.0min; 86 mg); octamer (48.9 min; 41 mg); nonamer (52.2 min; 22 mg);decamer (56.2 min; 13.5 mg); undecamer (61.4 min; 8.2 mg). All productsfrom the pentamer on required additional purification because of peaktailing, which led to a contamination with lower oligomers thatincreased with the degree of oligomerization, and because of increasingcontamination with unidentified aliphatic material from the nonpolarsolvent and/or column. For sample preparation, a small percentage oftetrahydrofuiran had to be added to the CH₃CN from the heptamer onbecause of limited solubility in CH₃CN alone. For the pentamer throughnonamer, the additional purification was performed on column D (0–30minutes, 80 to 100% CH₃CN in water, then CH₃CN). For the decamer andundecamer, column B was used in combination with the same gradient. Thenonamer, decamer, and undecamer still contained excessive amounts ofaliphatic impurities after this treatment and were subjected to a thirdHPLC purification on column A using the same gradient. The followingyields of pure products (97% or better by HPLC) were obtained: pentamer,987 mg (41%); hexamer, 226 mg (16%); heptamer, 68 mg (6.1%); octamer, 26mg (2.7%); nonamer, 11.5 mg (1.3%); decamer, 6.5 mg (0.8%); undecamer,2.5 mg (0.3%). Total mass balance relative to3-O-acetyl-4[(2-benzothiazoyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin:82%.

Example 10 Hydrolysis of Acetyl Protecting Groups from Acetyl- andBenzyl-Protected Oligomers

Part A—Tris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-Trimer

To a solution of 1.54 g (742 μmol) oftris(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer in30 mL of tetrahydrofuran was added all at once 5.8 mL (8.9 mmol) of 40%aqueous tetra-n-butylammonium hydroxide. The reaction mixture wasallowed to stand at room temperature in a closed flask for 94 hours,then partially evaporated to remove the tetrahydrofuran. The residue wasdiluted with 20 mL of water, the product was extracted twice with 20 mLethyl acetate, and the combined organic phases were washed with 10 mL ofbrine and evaporated. Filtration over a short silica gel column withethyl acetate yielded, after evaporation and drying in vacuo, 1.44 g(99%) of tris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer as acolorless foam.

Part B—Tetrakis(5,7,3′,4′-tetra-O-benzyl)eipicatechin (4β,8)₃-Tetramer

Reaction of 1.59 g (573 μmol) oftetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₃-tetramer with 5.6 mL (8.6 mmol) of 40% aqueoustetra-n-butyl-ammonium hydroxide in 29 mL of tetrahydrofuran for 96hours (as described for the trimer) yielded 1.45 g (97%) oftetrakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₃-tetramer as acolorless foam.

Part C—Pentakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₄-Pentamer

Reaction of 1.81 g (524 μmol) ofpentakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)-epicatechin(4β,8)₄-pentamer with 6.9 mL (10.5 mmol) of 40% aqueoustetra-n-butyl-ammonium hydroxide in 35 mL of tetrahydrofuran for 118hours, as described for the trimer, yielded 1.45 g (97%) of pentakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₄-pentamer as a colorlessfoam. The analytical sample was further purified by preparative HPLC(Column B, 0–30 min, 80–100% CH₃CN/H₂O, then CH₃CN.

Part D—Hexakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₅-Hexamer

Reaction of 486 mg (117 μmol) ofhexakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₅-hexamerwith 1.5 mL (2.3 mmol) of 40% aqueous tetra-n-butyl ammonium hydroxidein 8 mL of tetrahydrofuran for 101 hours (as described for the trimer)yielded 455 mg (100%) of hexakis(5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₅-hexamer as a colorless glass.

Part E—Heptakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₆-Heptamer

Reaction of 126 mg (26.1 μmol) ofheptakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₆-heptamer with 0.34 mL (0.52 mmol) of 40% aqueoustetra-n-butyl-ammonium hydroxide in 1.8 mL of tetrahydroftiran for 94hours (as described for the trimer) yielded 118 mg (100%) ofheptakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₆-heptamer as acolorless foam.

Part F—Octakis(5,7,3′, 4′-tetra-O-benzyl)epicatechin (4β,8)₇-Octamer

Reaction of 41.2 mg (26.1 μmol) ofoctakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₇-octamerwith 0.10 mL (0.15 mmol) of 40% aqueous tetra-n-butyl ammonium hydroxidein 0.5 mL of tetrahydrofuran for 126 hours (as described for the trimer)yielded 39.4 mg (102%) of octakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₇-octamer as a colorless foam.

Part G—Nonakis (5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₈-Nonamer

Reaction of 17.9 mg (2.88 μmol) of nonakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₈-nonamer with 47μL (72 μmol) of 40% aqueous tetra-n-butyl ammonium hydroxide in 0.3 mLof tetrahydrofuran for 134 hours (as described for the trimer) yielded16.8 mg (100%) of nonakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₈-nonamer as a colorless foam.

Example 11 Preparation of Epicatechin (4β,8)-Oligomers fromBenzyl-Protected Oligomers

A. Preparation of Epicatechin (4β,8)₂-Trimer

To a solution of 64.3 mg (33.0 μmol) ofbis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer in 5 mL oftetrahydrofuran were added 5 mL of methanol, 0.25 mL of water, and 57 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 80 minutes and filtered over cotton. The filtrationresidue was washed two times with 10 mL of methanol. The combinedfiltrates were evaporated, and the residue was taken up in 10 mL of HPLCgrade water. The solution was filtered and lyophilized to yield 32.4 mg(101%) of epicatechin (4β,8)₂-trimer 6H₂O as a fluffy, amorphous,off-white solid: [α]_(D)+70.40°, [α]₅₄₆+84.40° (MeOH, c 2.2 gL⁻¹); (ref.4c: [α]_(D)75.20°, acetone, c 8.7 gL⁻¹;ref. 4d: [α]₅₇₈+90°, MeOH, c 2gL⁻¹; ref. 6: [α]_(D)+76.4°, acetone, c 8.6 gL⁻¹; ref 19b: [α]₅₇₈+92°,H₂O c 1.9 gL⁻¹; ref. 19k: [α]_(D)+80°, MeOH, c 1.6 gL⁻¹); ¹³C NMR(CD₃OD, TMS; δ 60–85 region only) δ 79.73, 77.08, 73.47, 72.94, 66.84;MS (API/ES) m/z 865.0 (calcd for [M-H]⁻: 865.2), 577.0 (6%), 288.9 (4%).Analysis: Calculated for C₄₅H₃₈O₁₈.6H₂O: C, 55.44; H, 5.17. Found: C,55.71; H, 5.07.

B. Preparation of Epicatechin (4β,8)₃-Tetramer

To a solution of 56 mg (21.6 μmol) oftris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₃-tetramer in 4 mL oftetrahydrofuran were added 4 mL of methanol, 0.2 mL of water, and 47 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 75 minutes and filtered over cotton. The filtrationresidue was washed two times with 5 mL of methanol. The combinedfiltrates were diluted with 5 mL of HPLC grade water and partiallyevaporated to remove the organic solvents. After dilution with another10 mL of HPLC grade water, the solution was filtered and lyophilized toyield 24.4 mg (89%) of epicatechin (4β,8)₃-tetramer-6H₂O as a fluffy,amorphous, off-white solid: [α]_(D)+93.3°, [α]₅₄₆+114° (MeOH, c 9.3gL⁻¹) (ref. 4d: [α]₅₇₈+73.2°, MeOH, c 3.7 gL⁻¹; ref. 4j: [α]_(D)+59.8°,acetone, c 12 gL⁻¹; ref. 6: [α]_(D)+109.5°, acetone, c 12.3 gL⁻¹; ref.19i: [α]_(D)+89.2°, acetone, c 9 gL⁻¹; ref. 191: [α]_(D)+81°, MeOH, c1.1 gL⁻¹); MS (API/ES) m/z 1153.3 (55%; calcd for [M-H]⁻: 1153.3), 865.1(25%), 576.9 (100%), 500.1 (30%), 288.9 (4%). Analys: Calculated forC₆₀H₅₀O₂₄.6H₂O: C, 56.96; H, 5.10. Found: C, 56.98; H, 4.83.

C. Epicatechin (4β,8)₄-Pentamer

To a solution of 76 mg (23.4 μmol) ofpentakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₄-pentamer in 4 mLof tetrahydrofuran were added 4 mL of methanol, 0.2 mL of water, and 60mg of 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under1 bar of hydrogen for 2 hours and filtered over cotton. The filtrationresidue was washed with methanol, and the combined filtrates werepartially evaporated to remove the organic solvents. The residue wasdiluted with 10 mL of HPLC-grade water, filtered, and lyophilized toproduce 34.8 mg of epicatechin (4β,8)₄-pentamer as a fluffy, amorphous,off-white solid: [α]_(D)+116°, [α]₅₄₆+140° (methanol, c 8.3 gL⁻¹) (ref.4d: [α]₅₇₈+96°, meOH, c 1 gL⁻¹; ref. 19i: [α]_(D)+102.1°, acetone, c 10gL⁻¹; ref. 191: [α]_(D)+102°, MeOH, c 1.2 gL⁻¹). Analysis: Calculatedfor C₇₅H₆₂O₃₀.7.5H₂O: C, 57.07; H, 4.92. Found: C, 56.99; H, 4.79.

D. Preparation of Epicatechin (4β,8)₅-Hexamer

To a solution of 92.3 mg (23.7 μmol) ofhexakis(5,7,3′,4′tetra-O-benzyl)epicatechin (4β,8)₅-hexamer in 8 mL oftetrahydrofuran were added 8 mL of methanol, 0.4 mL of water, and 169 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 50 minutes and filtered over cotton. The filtrationresidue was washed with methanol, and the combined filtrates werepartially evaporated after addition of 10 mL of HPLC-grade water. Theresidue was diluted with another 20 mL of KPLC-grade water, filtered,and lyophilized to produce 47.4 mg of epicatechin (4β,8)-hexamer as afluffy, amorphous, off-white solid: [α]_(D)+123°, [α]₅₄₆+149°,(methanol, c 8.6 gL⁻¹). Analysis: Calculated for C₉₀H₇₄O₃₆.9.2H₂O: C,56.98; H, 4.91. Found: C, 56.89; H, 4.61.

E. Preparation of Epicatechin (4β,8)₆-Heptamer

To a solution of 87.5 mg (19.3 μmol) of heptakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₆-heptamer in 8 mL oftetrahydrofuron were added 8 mL of methanol, 0.4 mL of water, and 111 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen (H₂) for 1 hour and filtered over cotton. The filtrationresidue was washed with MeOH, and the combined filtrates were partiallyevaporated after addition of 10 mL of HPLC-grade H₂O. The residue wasdiluted with another 10 mL of HPLC-grade H₂O, filtered, and lyophilizedto produce 39.3 mg of epicatechin (4β,8)₆-heptamer as a fluffy,amorphous, off-white solid: [α]_(D)+134°, [α]₅₄₆+164° (MeOH, c 9.6gL⁻¹). Analysis: Calculated for C₁₀₅H₈₆O₄₂.10H₂O: C, 57.33; H, 4.86.Found: C, 57.49; H, 4.80

F. Preparation of Epicatechin (4β,8)₇-Octamer

To a solution of 35.7 mg (6.88 μmol) of octakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₇-octamer in 3 mL oftetrahydrofuran were added 3 mL of methanol, 0.15 mL of water, and 57 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 55 minutes and filtered over cotton. The filtrationresidue was washed with methanol, and the combined filtrates werepartially evaporated after addition of 10 mL of HPLC-grade water. Theresidue was diluted with another 10 mL of HPLC-grade water, filtered,and lyophilized to produce 17.1 mg of epicatechin (4β,8)₇-octamer as afluffy, amorphous, off-white solid: [α]_(D)+148°, [α]₅₄₆+180° (methanol,c 5.2 gL⁻¹). Analysis: Calculated for C₁₂₀H₉₈O₄₈.10.7H₂O: C, 57.66; H,4.77. Found: C, 57.68; H, 4.79.

The HPLC analyses of the purified natural and synthetic oligomers werecompared. The purified natural oligomers all exhibited additional peaks,with the number of additional peaks increasing as the oligomeric sizeincreased.

Example 12 Self-Condensation of4-[(2-Benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin Inducedby Silver Tetrafluoroborate

To a solution of 445 mg (545 μmol) of4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin (themajor diastereoisomer of Example 4) in 5 mL of anhydrous tetrahydrofuranwas added dropwise within 30 minutes in dim light and with magneticstirring and ice cooling a solution of 48 mg (247 μmol) of silvertetrafluoroborate (dried at 100° C. in an oil pump vacuum with exclusionof light for 110 minutes immediately before use). Stirring at 0° C. wascontinued for 5 minutes, then 0.2 mL of triethylamine was added. Afterevaporation, the residue was prepurified by filtration over a shortsilica gel column with ethyl acetate/hexane (1:1) to yield 414 mg of acolorless foam. The five least polar major components of this complexmixture were isolated by preparative HPLC (column D; 0–30 minutes, 80 to100% methyl cyanide (CH₃CN) in water, then CH₃CN. The followingretention times and yields were observed: 2-mercaptobenzothiazole, t_(R)4.4 minutes, 19 mg;5,7,3′,4′-tetra-O-benzyl-4-(2-thioxobenzothiazol-3-yl)epicatechin, 15.4minutes, 18 mg (4%); starting monomer, 21.4 minutes, 14 mg (3%recovery);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[5,7,3′,4′-tetra-O-benzyl-4-(2-thioxobenzothiazol-3-yl)epicatechin],23.5 minutes, 7 mg (2%);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin],27.0 minutes, 15 mg (4%).

Example 13 Self-Condensation of4-[(2-Benzothiazolyl)thiol-5,7,3′,4′-tetra-O-benzyl-epicatechin Inducedby Acidic Clay

To a solution of 18.0 mg (21.0 μmol) of 4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin (major diastereoisomer inExample 4) in 1 mL of anhydrous methyl chloride (CH₂Cl₂) was added 38 mgof montmorrillonite clay sold under the tradename Bentonite K-10. Themixture was stirred at room temperature for 160 minutes, filtered, andevaporated. The residue was separated by preparative HPLC (column B;0–30 minutes, 80 to 100% CH₃CN in water, then CH₃CN. The followingretention times and yields were observed: 2-mercaptobenzothiazole, t_(R)4.6 minutes, 0.6 mg;5,7,3′,4′tetra-O-benzyl-4-(2-thioxobenzothiazol-3-yl)epicatechin, 13.2minutes, 2.0 mg (11%); starting monomer, 19.2 minutes, 2.7 mg (15%recovery);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[5,7,3′,4′-tetra-O-benzyl-4-(2-thixobenzothiazoll-3-yl)epicatechin,21.5 minutes, 0.6 mg (4%);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[4-((2-benzothiazol)thio)-5,7,3′,4′-tetra-O-benzylepicatechin],25.9 minutes, 1.4 mg (9%);5,7,3′,4′-tetra-O-benzylepicatechin-4β,8)-(5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[4-((benzothiazothiazoyl)thio)-5,7,3′,4′-bistetra-O-benzylepicatechin], 30.8 minutes, 1.2 mg (8%);5,7,3′,4′-tetra-O-benzylepicatechin-bis(4β,8)-(5,7,3′,4′-tetra-O-benzlepicatechin]-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin],34.2 minutes, 0.3 mg (2%).

Example 14 Self-Condensation of3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinInduced by Silver Tetrafluoroborate

To a solution of 355 mg (414 μmol) of3-O-acetyl-4-[(2-benzathiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin4 mL of anhydrous tetrahydrofuran was added dropwise within 40 minutesin dim light and with magnetic stirring and ice cooling a solution of 20mg (103 μmol) of silver tetrafluoroborate dried at 90° C. in an oil pumpvacuum with exclusion of light for 1 hour immediately before use.Stirring at 0° C. was continued for 10 minutes, then 0.2 mL oftriethylamine was added. After evaporation, the residue was prepurifiedby filtration over a short silica gel column with ethyl acetate/hexane(1:1) to yield 331 mg of a colorless foam. This mixture was separated bypreparative HPLC (column D; 0–30 minutes, 80 to 100% methyl cyanide(CH₃CN) in water, then CH₃CN). The following retention times and yieldswere observed: 2-mercaptobenzothiazole, t_(R) 4.6 min, 6.4 mg;3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin, 11.1 min.,13.2 mg (4.5%);3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin),16.9 min, 38.3 mg (13%); starting material, 22.4 min., 156 mg. (44%); amixture of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]and3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin):29.7 min, 54.3 mg; a mixture of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]and3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin):34.6 min., 11.9 mg;3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4-tetra-O-benzyl)epicatechin]-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]:38.9 min., 6.3 mg (2.1%). The mixture of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]and3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)was separated by normal-phase HPLC (Column F; 0–40 min, 20 to 50% ethylacetate (EtOAc) in hexane, then 50%) to yield 43 mg (14%) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin](t_(R) 22.1 min) and 6.4 mg (2.2%) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-4β,8-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)(tR 32.8 min.). The mixture of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]and3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4hydroxyepicatechin) was separated on column E using the same gradient toyield 5.4 mg (1.8%) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin](t_(R) 34.8 min) and 5.0 mg (1.7%) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)(t_(R) 42.8 min). For characterization, the products obtained by normalphase HPLC were repurified on column B (0–30 min, 80 to 100% methylcyanide (CH₃CN) in water then CH₃,CN).

Example 15 Reaction of3-O-Acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-4β,8-[3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin]with Tetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)(4β,8)₃-Tetramer

A 21 mg (0.1 1 mmol) sample of silver tetrafluoroborate was dried in thereaction flask at 100° C. in an oil pump vacuum with exclusion of lightfor 1 hour. After cooling, the vacuum was broken with nitrogen, and asolution of 190 mg (68.8 μmol) of tetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₃-tetramer in 1mL of anhydrous tetrahydrofuran was added all at once. The flask wasplaced in an ice bath under dim light, and a solution of 35.5 mg (22.9μmol) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin)in 0.5 mL of anhydrous tetrahydroftiran was added dropwise in 12 minuteswith stirring. Stirring was continued for 5 minutes at 0° C. and for 10minutes at room temperature. Triethylamine (0.1 mL) was added, themixture was evaporated, and the residue was filtered over a short silicagel column with ethyl acetate/hexane (1:1). The eluate was evaporated,and the crude product mixture (230 mg) was separated by preparative HPLC(column D, 280 nm; 0–30 min, 80 to 100% methyl cyanide (CH₃CN) in water,then CH₃CN. The following retention times and yields were observed:3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin),t_(R) 22.7 min., 21.0 mg (65%);3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin),34.6 min., 0.8 mg (2.5%);tetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₃-tetramer, 36.3 min., 176 mg (92.5% recovery);hexakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₅-hexamer,45.6 min., 11.7 mg (12%);octakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₇-octamer,1.4 mg (2.2%).

Example 16 Anticancer Activity

Cell cycle analysis of procyanidin-treated MDA MB-231 human breastcancer cells showed a G₀/G₁ arrest by the pentamer, no effect by thedimer or trimer, and only a slight effect by the tetramer (See Table 1).

TABLE 1 Cell Cycle Analysis of MDA MB-231 Human Breast Cancer CellsTreated with Oligomeric Procyanidins Purified from Cocoa % G₀/G₁ % S %G₂/M Control 36.69 23.39 39.92 Vehicle 38.26 22.43 39.30 Dimer (200μg/mL; 24 hrs) 38.13 22.43 39.45 Control 42.28 35.61 22.12 Vehicle 43.6034.10 22.30 Trimer (200 μg/mL; 24 hrs) 43.22 35.98 20.80 Control 40.3336.25 23.42 Vehicle 43.71 34.42 21.87 Tetramer (200 μg/mL; 24 hrs) 51.4628.25 20.30 Control 38.33 21.05 40.61 Vehicle 37.84 21.39 40.77 Pentamer(200 μg/mL; 24 hr) 66.03 17.23 16.67 Pentamer (200 μg/mL; 48 hr) 88.316.07 5.62The increase in G₀/G₁ was accompanied by a decrease of cell numbers inthe S phase and in the G₂/M phase.

The manner of cell death (apoptosis or necrosis) was investigated by theannexin V-fluorescein isothiocyanate (FITC) assay using Trevigen's TACS™Annexin V-FITC kit. Cell cycle analysis of MDA MB-231 cells treated withnatural and synthetic procyanidin trimer, tetramer, and pentamer isshown below. Flow cytometry of procyanidin-tested MDA MD 231 humanbreast cover cells using annexin V-FITC and propidium iodide (controlversus 24 hour treatment with 200 μg/mL of oligomer) is shown below. Ais the epicatechin (4β,8)₂-trimer. B is the epicatechin(4β,8)₃-tetramer. C is the epicatechin (4β,8)₄-pentamer. The lower leftquadrant shows viable cells. The lower right quadrant shows earlyapoptic events. The upper right quadrant shows late apoptic events. Theupper left quadrant shows nonviable cells.

Cells treated with both natural and synthetic procyanidins showedsimilar profiles, with increases in cell populations in the upper rightquadrant being observed as the oligomer size increased. This quadrantrepresents annexin V positive cells that also take up propidium iodide,which cells are considered to be in either late apoptosis or innecrosis. The absence of a distinct cell population in the lower rightquadrant (cells associated with early apoptotic events) in the case ofpentamer-treated cells suggests a necrotic pathway to cell deathpossibly due to a direct interaction with the cell membrane leading todamage, cell crisis, and eventual death.

The pentamer-caused G₀/G₁ arrest was reversible in cells treated up to 8hours and irreversible after a 24 hour treatment. No difference inactivity was observed between natural and synthetic procyanidin trimer.An approximately 15% increase in G₀/G₁ arrest was seen for syntheticprocyanidin tetramer compared to natural procyanidin tetramer. Anapproximately 30% increase for synthetic vs. natural procyanidinpentamer was shown. See Table 2 below.

TABLE 2 Comparison Cell Cycle Analysis of MDA MB-231 Human Breast CancerCells Treated with Natural versus Synthetic Oligomeric Procyanidins %G₀/G₁ % S % G₂/M Control 28.65 49.28 22.06 Vehicle 27.19 49.61 23.2Natural trimer (200 μg/mL; 24 hr) 28.46 48.49 23.05 Synthetic trimer(200 μg/mL; 24 hr) 26.98 49.57 23.45 Natural tetramer (200 μg/mL; 24 hr)36.82 43.37 19.02 Synthetic tetramer (200 μg/mL; 24 hr) 43.49 39.3917.03 Natural pentamer (200 μg/mL; 24 hr) 45.99 38.25 15.76 Syntheticpentamer (200 μg/mL; 24 hr) 64.15 23.36 12.49

A recent report indicated that hydrogen peroxide (H₂O₂) wasartifactually produced in vitro by several different polyphenoliccompounds and was responsible for causing a variety of biologicalactivities. See Long, L. H. et al., Biochem. Biophys. Res. Commun.,2000, 273, 50. The results in Table 3 show that if hydrogen peroxide waspresent at the levels reported in the literature, it would produce ashift in the cell cycle to G₂/M with a decrease in G₀/G₁. The additionof catalase abrogated these effects, causing a shift in the cell cycleback to control values. The addition of catalase alone topentamer-treated cells produced no conclusive change in the cell cycleattributable to hydrogen peroxide, i. e., the typical G₀/G₁ arrestcaused by the pentamer remained essentially unchanged.

To eliminate the possibility that the epicatechin (4β,8)₄-pentamer mightinhibit catalase activity, hydrogen peroxide was added topentamer-treated cells in the presence and absence of catalase. Theaddition of hydrogen peroxide to pentamer-treated cells led to anincrease in G₀/G₁ and G₂/M arrest at the expense of cells in the Sphase. Catalase addition caused a shift back to the G₀/G₁ arrest typicalof pentamer-treated cells, and heat-inactivated catalase had no effect.Thus, the G₀/G₁ arrest was directly caused by the pentamer, not by thehydrogen peroxide. These differences can be attributed to the higherpurities of the synthetic procyanidins.

TABLE 3 Cell Cycle Analysis of MDA MB-231 Pentamer Treated Cells % G₀/G₁% S % G₂/M Control 33.14 44.06 22.81 Vehicle 36.44 41.63 21.94 100 μMH₂O₂; 24 hr 20.32 44.92 34.76 100 μM H₂O₂ + catalase; 24 hr 35.20 42.9321.86 100 μM H₂O₂ + heat inactivated catalase; 20.27 45.48 34.25 24 hrControl 29.87 46.21 23.92 Vehicle 30.28 47.25 22.47 Pentamers (200μg/mL); 24 hr 44.94 38.01 17.05 Pentamers (200 μg/mL) + catalase; 24 hr41.23 39.65 20.12 Pentamers (200 μg/mL) + heat inactivated 42.89 39.4317.68 catalase; 24 hr Pentamers (200 μg/mL) + 100 μM H₂O₂; 42.67 18.6338.71 24 hr Pentamers (200 μg/mL) + 100 μM H₂O₂ + 48.20 31.12 20.68catalase; 24 hr Pentamers (200 μg/mL) + 100 μM H₂O₂ + 39.47 23.39 37.14heat inactivated catalase; 24 hr

Collectively, the above results confirm the cytotoxicity to human breastcancer cell lines by an epicatechin pentamer, whether purified fromcocoa polyphenol extracts or prepared synthetically. The procyanidinpentamer caused a G₀/G₁ arrest in MDA MB-231 cells which was independentof any effects caused by hydrogen peroxide. An increase in annexin V andpropidium iodide positive cells suggests that the pentamer-treated cellsquickly entered into a necrotic phase of cell death.

The above examples are merely illustrative and no limitation of thepreferred embodiments is implied. The skilled artisan will recognizemany variations without departing from the spirit and scope of theinvention.

1. A process for preparing a 5,7,3′,4′-tetra-O-protectedprocyanidin-(4,8)-dimer comprises coupling a5,7,3′,4′-tetra-O-protected-epicatechin or -catechin monomer with a5,7,3′,4′-tetra-O-protected-4-alkoxy-epicatechin or -catechin monomer inthe presence of an acidic clay.
 2. The process of claim 1, wherein theprotecting groups are groups which do not deactivate the A ring of theprotected monomers; wherein the 4-alkoxy group is a C₂–C₆ alkoxy grouphaving a terminal hydroxy group; and wherein the acidic clay is aBentonite clay.
 3. The process of claim 1, wherein the5,7,3′,4′-tetra-O-protected monomer is5,7,3′,4′-tetra-O-benzyl-epicatechin; wherein the5,7,3′,4′-tetra-O-benzyl-protected-4-alkoxy monomer is5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-epicatechin; and whereinthe protected dimer isbis(5,7,3′,4′-tetra-O-benzyl)-epicatechin-(4β,8)-dimer.
 4. The processof claim 3, wherein the yield of the insolated dimer is about 90%. 5.The process of claim 1, wherein the coupled monomers are5,7,3′,4′-tetra-O-benzyl-epicatechin and5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-epicatechin and wherein theprotected dimer is5,7,3′,4′-tetra-O-benzyl-epicatechin-(4β,8)-5,7,3′,4′-tetra-O-benzyl-epicatechin;wherein the coupled monomers are 5,7,3′,4′-tetra-O-benzyl-epicatechinand 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-catechin and whereinthe protected dimer is5,7,3′,4′-tetra-O-benzyl-(4β,8)-5,7,3′,4′-tetra-O-benzyl-catechin; andwherein the coupled monomers are 5,7,3′,4′-tetra-O-benzyl-catechin and5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-catechin; and wherein theprotected dimer is5,7,3′,4′-tetra-O-benzyl-catechin-(4α,8)-5,7,3′,4′-tetra-O-benzyl-catechin.